The invention relates to a system for delivering genetic material into cells in situ in a patient.
Various methods for introducing genetic material into an internal or external target site on an animal exist. Most prominent are methods of accelerated particle mediated gene transfer, such as are described in U.S. Pat. No. 4,945,050 (Sanford, et al.), U.S. Pat. No. 5,204,253 (Sanford, et al.) and U.S. Pat. No. 5,015,580 (Christou, et al.).
Other methods have focussed on introducing genetic material into skin cells, particularly keratinocytes. Keratinocytes are the principle cells which cover the surface of the body. They are capable of producing proteins, particularly keratin, which constitute the main surface barriers of the body. For several different reasons, keratinocytes are attractive potential targets for gene transfer. Since they are located on the surface of the body, they are easily accessed both for gene manipulation and monitoring. If complications from gene transfer would occur, for instance, the development of local tumors or local infections, these could more easily be treated in the skin than elsewhere.
To date, genetic manipulation of keratinocytes has been done in one principal way. Skin has been harvested, the keratinocytes have been separated from the fibroblasts, and then the keratinocytes individually isolated and brought into suspension. These suspensions of keratinocytes have then been cultured to confluence using tissue culture techniques, as reported by Rheinwald, J. G., Green, H. Serial Cultivation of Human Epidermal Keratinocytes: The Formation of Keratinizing Colonies From Cells. Cell G, 331-343, 1975.
The new genetic material has been introduced into the keratinocyte while being grown in vitro using either a viral vector or plasmid, as reported by Morgan, J. R., Barrandon, Y., Green, H., Mulligan, R. C. Expression of an Exogenous Growth Hormone Glue by Transplantable Human Epidermal Cells. Science, Vol. 237, 1476-1479 (1987) and Tenmer, J., Lindahl, A., Green, H. Human Growth Hormone in the Blood of Athymic Mice Grafted With Cultures of Hormone-Secreting Human Keratinocytes. FASEB J., 4:3245-3250 (1990). The transfected cells are then usually resuspended and grown on selective media in order to increase the yield of transfection. Sheets of keratinocytes are then transplanted back to the mammal from which the keratinocytes were harvested.
Even though the in vitro yield has been acceptable, the in vivo yield has been unacceptably low, both short and long term. It has been very difficult to document any significant long term (more than thirty days) expression with these techniques, for example, as reported by Garlick, J. A., Katz, A. B., Fenvjes Esitaichman, L. B. Retrovirus Mediated Transduction of Cultured Epidermal Keratinocytes. J. Invest. Dermatol., 97:824-829, 1991.